Evaluation of soil bacteria as bioinoculants for the control of field pea root rot caused by Aphanomyces euteiches A Thesis Submitted to the College of Graduate and Postdoctoral Studies in Partial Fulfilment of the Requirements for the Degree of Master of Science in the Department of Soil Science University of Saskatchewan Saskatoon By Ashebir Tsedeke Godebo © Copyright Ashebir Tsedeke Godebo, March 2019. All rights reserved. PERMISSION TO USE In presenting this thesis in partial fulfilment of the requirement for a post graduate degree from the University of Saskatchewan, I agree that the library of this University may take it freely available for inspection. I further agree that permission for copying of this thesis in any manner, in whole or in part, for scholarly purpose may be granted by the professor or professors who supervised my thesis work or, in their absence, by the Head of the Department or the Dean of the College in which my thesis work is done. It is understood that any copying or publication or use of this thesis part or its parts for financial gain shall not be allowed without my written permission. It is also understood that due consideration shall be given to me and to the University of Saskatchewan in any scholarly use which may be made of any material in my thesis. Request for permission to copy or to make other use of any material in this thesis in whole or part should be addressed to: Head of the Department of Soil Science 51 Campus drive University of Saskatchewan Saskatoon, Saskatchewan S7N 5A8 Canada OR Dean College of Graduate and Postdoctoral Studies University of Saskatchewan 116 Thorvaldson Building, 110 Science Place Saskatoon, Saskatchewan S7N 5C9 Canada i DISCLAIMER Reference in this thesis to any specific commercial product, process, or service by trade name, trademark, manufacturer, or otherwise, does not constitute or imply its endorsement, recommendation, or favoring by the University of Saskatchewan. The views and opinions of the author expressed herein do not state or reflect those of the University of Saskatchewan and shall not be used for advertising or product endorsement purposes. ii ABSTRACT Aphanomyces euteiches is an oomycete pathogen that is becoming a serious problem for field pea (Pisum sativum L) production in western Canada. The pathogen causes severe rot in the root, cortex, and epicotyl of field pea resulting in stunting, yellow and wilting leaves, or plant death. Aphanomyces root rot develops because of zoosporic or myceliogenic infection when oospores germinate to form germ sporangia and germ tubes, respectively. Until the recent introduction of the fungicide INTEGOTM Solo (ethaboxam) and Vibrance® Maxx RFC, there was no fungicide available in Canada that effectively suppresses or controls aphanomyces root rot in field pea. Additional control measures are needed, and one addition to chemical control is the development of bacterial inoculants that interrupt the pathogen’s lifecycle and ultimately control or reduces disease expression in the host plant. This study comprised laboratory studies aimed at isolating and identifying antagonistic bacteria against A. euteiches mycelia and zoospore growth stages, and growth chamber trials that examined efficacy of antagonistic bacteria as biocontrol agents against aphanomyces root rot in field pea. Soils were collected from 43 commercial field pea fields across Saskatchewan. Initial screening of antagonistic bacteria was completed by assessing mycelia growth inhibition of A. euteiches in vitro. Growth inhibition of each antagonistic bacterium was further evaluated using a dual plate assay technique where single colonies of the antagonistic bacterial isolates were inoculated at two opposite edges on a PDA plate and a plug of A. euteiches mycelia was placed in the center of the plate. Additionally, a preliminary screening assay utilizing a dual plate technique was employed to assess 170 bacterial isolates for biocontrol activity against A. euteiches. These iii 170 bacterial isolates were from a previously existing bacteria culture collection of roots associated rhizobacteria from a variety of field crops. The antagonistic bacterial isolates were also assayed for in vitro zoospore germination inhibition. A total of 184 antagonistic bacteria, of which 22 were from a previously existing bacteria culture collection, that inhibited the mycelia stage of A. euteiches were identified using the initial screening assay. Of these, 47 inhibited zoospore germination by 75% or more compared to a control assay plate. The mean mycelial growth inhibition potential of the isolates ranged from 1 mm to 12 mm whereas the mean zoospore germination inhibition potential of the isolates ranged from 0 to 100 %. Based on 16S rDNA gene sequencing, the antagonistic bacterial isolates were placed into 18 different genera with Bacillus, Pseudomonas, Paenibacillus, Lysobacter and Streptomyces being the top five containing 45, 32, 29,17 and 12 antagonistic bacterial isolates, respectively. Bacterial isolates that inhibited mycelia growth and zoospore germination by 75% or more were selected for further evaluation in growth chamber trials. In the first set of experiments (Trial 1) pea plants were grown in vermiculite and inoculated with antagonistic bacterial suspensions and A. euteiches zoospores. Four weeks after planting, the pea plants were harvested, and roots assessed for level of disease development. Isolates which significantly suppressed aphanomyces root rot in vermiculite were further evaluated as soil inoculants in pot experiments using non- sterile field soil (Trial 2). Screening of 47 antagonistic bacteria as bioinoculants in growth chamber Trial 1 identified 29 that significantly (α = 0.05) suppressed or reduced aphanomyces root rot in field pea. Of these, 20 isolates were selected and screened as soil inoculants in a second set of experiments (Trial 2) iv and three isolates produced the highest biocontrol activity and significantly (α = 0.05) suppressed or reduced aphanomyces root rot in field pea. From the findings of this research, it can be concluded that the A. euteiches lifecycle can be interrupted using rhizosphere bacteria and hence these bacterial isolates may be used as biocontrol agents to suppress or reduce aphanomyces root rot in field pea. Variations of inhibition potential among isolates suggests that the mechanisms by which biocontrol is achieved such as the production and secretion of inhibitory compounds and/or the mode of action exerted by the inhibitory metabolites likely varies among isolates. The results of this research indicate the potential promise for the development of microbial biocontrol agents. Further studies aimed at assessing the efficacy of the promising isolates under field conditions in Saskatchewan and other manipulative studies that would maximize biocontrol potential and their effective utilization are a necessary next step. v ACKNOWLEDGMENTS I am truly thankful to my supervisors Drs. Fran Walley and Jim Germida for their continuous help and support throughout my program at the University of Saskatchewan. Without their guidance and help, I wouldn’t have been able to excel in my studies and research. I would also like to thank and greatly appreciate the time and help given by other committee members, Drs. Sabine Banniza and Jeff Schoenau. I am grateful to Dr. Bobbi Helgeson and people in Soil Microbiology lab. A very special thanks go to Jorge Cordero, Eduardo Kovalski, Akeem Shorunke, Claire Kohout, Zayda Morales, Bethany Templeton, Panchali Katalunda and the summer students Ian Hnatowich and Kyler Kanegawa. This also extends to Nimllash Sivachandrakumar and Laura Cox from Dr. Sabine’s lab who provided technical help. I would also like to thank Marc St. Arnaud and Kim Heidinger from the Department of Soil Science for the administrative assistance. Finally, I would like to thank the Saskatchewan Pulse Growers for supporting this project financially. vi TABLE OF CONTENTS PERMISSION TO USE ................................................................................................................................. i DISCLAIMER .............................................................................................................................................. ii ABSTRACT ................................................................................................................................................. iii ACKNOWLEDGMENTS ........................................................................................................................... vi TABLE OF CONTENTS ............................................................................................................................ vii LIST OF TABLES ........................................................................................................................................ x LIST OF FIGURES .................................................................................................................................... xii LIST OF EQUATIONS ............................................................................................................................. xiv 1. GENERAL INTRODUCTION ............................................................................................................. 1 1.1 GENERAL OBJECTIVE .............................................................................................................. 4 1.1.1 Specific objectives ...............................................................................................................